Amaresh Kumar Ranjan,
Mugdha V. Joglekar,
Ashwini N. Atre,
Milind Patole,
Ramesh R. Bhonde,
*
Anandwardhan A. Hardikar
Pages 949 - 953
Keywords:
immunofluorescence,
in situ PCR,
miRNA,
protein,
single cell,
transcript
Abstract:
Cellular mechanisms that inhibit mRNA translation by regulatory molecules involving microRNAs (miRNAs), a class of noncoding RNAs (ncRNAs), are well recognized in recent days. However, methodologies that measure these changes in cell populations lack the capabilities to observe such effects at single cell resolution. This is mostly due to the low level of transcript abundance and the heterogeneity of cell populations, together with the inability to measure transcripts and proteins at the same time. Here, we combine an in situ TaqMan PCR method with immunostaining so as to amplify low abundance transcripts in cellular compartments and image these efficiently at single cell resolution. The method offers flexibility to end-users for further fine-tuning of this optimized protocol based on the number of PCR cycles for individual genes in any cell type. After immunostaining, confocal microscopy is performed to detect the fluorescence of TaqMan probes (representing amplified transcripts/miRNA) and fluorophores tagged to antibodies (representing proteins) simultaneously. The presented technique offers an important tool to understand functional genomics as well as molecular mechanism of transcriptional and translational regulation so as to map these at single cell resolution.
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